lecture 7d- chromatography (1h) (2)

8/11/2019 Lecture 7d- Chromatography (1h) (2)

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USTH

Analytical chemistry

Lecture 7d:

Chromatography



7.1. Introduction to

Chromatography



ANALYTICAL SEPARATIONS

• Challenge: qualitative and quantitative information

about several substances (analyte) present in thesample.

- Mixture: a multi-analyte or multi-component sample.

• - The components of the sample that are not of our

interest constitute a sample matr ix .• The analysis of a mixture can be achieved in two

ways:

o a selective detection

o a separation of the original sample into individualcomponents and the determination of these

components using simpler and cheaper analyticalmethods.

• The second a roach separat ion science



Classical separation methods• There are several methods of

separation, or isolation, based upondifferent physico-chemical principles:

- selective extraction,

- selective adsorption,- fractional crystallization,

- selective precipitation,

- selective chemical reactions,- electrophoresis (capitalizes on differences

in migrat ion rates in electric field),

- mass spectrometry.



Types of Chromatography

• Columnar vs. Planar Chromatography (TCL,paper chromatography)

• Contemporary Column Chromatography

Types

1. Gas Chrom. (GC); Gas/Liquid phases

2. Liquid Chrom. (LC); Liquid/Bonded

phases

3. Supercritical-Fluid Chrom. (SFC);SF/Bonded phases



MOBILE PHASE LIQUID

Liquid-Liquid

Chromatography

(Partition)

Liquid-Solid

Chromatography

(Adsorption)

Liquid Solid

Normal Phase Reverse Phase Normal Phase Reverse Phase

Mobile Phase -

Nonpolar

Stationary phase -

Polar

Mobile Phase -

Polar

Stationary phase -

Nonpolar

FORMAT

STATIONARY

PHASE

Types of Chromatography



Column chromatography• In column chromatography, a sample is dissolved in

a mobi le phase that moves along a co lumn

containing a stationary phase .

• The process of moving the sample through thecolumn is called elut ion .

• Sample interact with the stationary phase when the

sample moves along the column.

• Some components interact more strongly with thestationary phase than the other.

• The rate of m igrat ion along the column varies,

some components are retained longer in the columnthat other.



Column chromatography (con’t)

Individual components appear at the column outlet(or “are eluted”) with characteristic retent iont imes .

All of the components are detected at the outlet ofthe chromatographic column using a suitabledetector.

The signal from the detector is plotted vs. time andthe resulting chromatogram is recorded. Theinstrument in which a complete chromatographicanalysis is performed is called a

chromatograph .



CLASSIFICATION OF CHROMATOGRAPHY(with respect to the type of a mobile phase)

• gas chromatography - GC (good for volatileand thermally stable samples, a carrier gas

plus a gaseous sample)• liquid chromatography - LC, usually highperformance liquid chromatography, HPLC

A) normal phase HPLC

(polar stationary phase and non-polar solvents),B) reversed phase HPLC

(non-polar stationary phase and polar solvents).

• supercritical fluid chromatography - SFC



CLASSIFICATION OF CHROMATOGRAPHY (the type of interactions between the sample component

and the stationary phase)

• partition chromatography (an extraction principle);gas-liquid chromatography (GLC) and also in HPLC,• adsorption chromatography (adsorption isotherms);

gas-solid chromatography (GSC) and also in HPLC,• ion-exchange chromatography,

Two next steps: -A block diagram of a chromatograph-Chromatogram: a product of chromatographicanalysis, a plot of the signal from the chromatographic

detector vs. retention time (or volume).



A chromatogram: displaying basic parameters of

chromatographic peaks.



A frequent approximation of the peak area:

Retention times and relative retentions provide the basis for

the qualitative analysis.

The peak height hi or the peak area Ai, are the basis ofquantitative chromatographic analysis.

The relative retention:

If t’ R,A > t’ R,B, > 1 (the substance A is eluted later)

The higher , the better the separation (resolution) betweenthe two components A and B.

is practically independent of the flow rate: useful in

identifying peaks when flow rate changes.

i2/1ii WhA

A B

t R A

t R B

,,'

,'



RESOLUTION

We need to avoid overlapping of adjacent peaks for the quantitativeanalysis.

The quantitative measure of a separation quality is the resolution :

where: tR,A > tR,B

Wav is the average width of the neighboring peaks,

The selectivity , (tR,A - tR,B) is the ability of the stationary phase to displaya different strength of interaction with different analytes.

Efficiency , the column ability to produce narrow chromatographic peaks:the higher the efficiency the narrower the bands.

R s

t R A

t R B

W A

W B

t R A

t R B

W av

2(, ,

) (, ,

)

W av

W A

W B

2



Efficiency of a column is measured by a number of

theoretical plates (N), where N is:

L is an active length of a column and H (or HETP) is a “heightequivalent to a theoretical plate,” (or, a width of thehypothetical “slice”).

The higher N, the higher the efficiency.

The theoretical plate is a hypothetical “slice” of a columnwithin which an equilibrium of partition (adsorption) of ananalyte between the mobile and stationary phase is achieved.

N L

H



An expression relating chromatographic resolution to thenumber of theoretical plates, N, (efficiency) is:

where:

where K A, K B are partition coefficients of analytes A and B, respectively,and k’ A, k’ B, are the analyte capacity factors :

(The longer the component is retained in the column,the greater its capacity factor.)

R s N A B

A B

k Ak A

41

1

,

,

'

'

A B

k A

k B

K A

K B

,

'

'

k i

t R i

t M

t M

, ,



Number of theoretical plates is easily calculated from theexpression:

where s A is the standard deviation for peak A, expressed in

units of time.

N L

H

t R A

s A

,2

2



Height equivalent to a theoretical plate may be related to column internal “structure” and the average linear flow rate (u) of the mobile phase.

Van Deemter equation (packed columns, GC; there are other equationslike Snyder-Soczewinski for LC, Golay for CGC):

Terms A, B and C correspond to the following phenomena:

A - multiple paths effects (eddy diffusion),B - longitudinal diffusion,C - incomplete equilibration (non-equilibrium conditions) or

transport resistance

The more uniform are stationary phase (solid support) particles, the

lower the A. In the case of open tubular columns (WCOT) there is no Aat all (A = 0).

In the case of capillary electrophoresis, there is no equilibrium betweenthe two phases (migration is caused by another effect) so the onlyremaining term is longitudinal diffusion.

H A Bu

C u



Constants A, B and C can be calculated from physico-chemical parametersof the stationary phase, carrier gas, etc.

Extra-columnar effects also add to the band broadening (broadeningoutside the column), which include injectors (supposed to deliver a Dirac

delta profile), detectors and any other “dead volumes” between the twoinjectors and the detector.

For the purposes of identification of the analytes, so called retentionindexes (Kovats indexes) are used (in addition to relative retentions):

where: t’ R,Cn+a > t’ R,A > t’ R,Cn and n and n+a are number of carbon

atoms in two members of n-alkane homologous series “embracing”compound A.

I n a t t

t t A

A R Cn

R Cn a R Cn

100 log log

log log

' ,'

,'

,'



How to Improve Column

Performance

1. Increase L (but tr increases as well)

2. Decrease dp (but back pressure

increases dramatically)

3. Decrease diameter of column (but

loading capacity also decreases)

4. Optimize k’ (but RS increases only

slightly when k’>5)



MAIN DETECTORS IN GC

1. A thermal conductivity detector (TCD)

Detecting via a difference in the thermal conductivity of gaseous samples.

TCD detects a difference of thermal conductivity of pure carriergas and its mixture with an analyte. H2 or He are mainly usedas carriers, as they are characterized by an extremely highthermal conductivity.

It is a universal detector, detects everything which is not acarrier gas, but is lacking sensitivity.

The signal is the current necessary to balance a Wheatstonebridge led to imbalance by the presence of an analyte in oneof its chambers (the resistor is less cooled due to lowered

thermal conductivity). The electric current constitutes a signal.



Schematic diagram of Thermal Conductivity Detector (TCD).If R1 : R3 = R2 : R4, then signal (current) is = 0.



2. A flame ionization detector (FID).

Detecting via a difference in the extent of ionization in a high temperature of a

flame (in a “cool” plasma).

There is a miniature H2-air burner equipped with two electrodes at the endof a GC column. Ions are produced from organic molecules in the burnerand these are collected on the cathode. The ionic current constitutes asignal. FID is considered a universal detector for organic compounds.

Other important detectors in GC are:

- Electron Capture Detector (ECD)

- Photo Ionization Detector (PID),

- Flame Photometric Detector (FPD).

Schematic diagram of Flame Ionization Detector (FID).

DETECTORS IN HPLC



DETECTORS IN HPLC

• Refractive index detector.

• Fixed length UV cell detector (usually 254nm).• Diode array UV-visible detector.• Electrochemical detectors, e.g., thin layeramperometric detector.

Both GC and HPLC have been coupled with methodslike MS, FTIR, NMR, etc., where the latter ones serve

as highly selective detectors and can be utilized forboth qualitative and quantitative analysis. Suchcombinations are often named “hyphenatedmethods”.



Flow Cell for

UV-Vis Detection



Diode Array UV-VIS Detector



Anthracene

detected at 250 nmwith Diode Array



Quantitative chromatographic



Quantitative chromatographic

analysis

• The detector can produce different peak areas

for the same amounts of two substances.

Sensitivity of the detector (or the detector

response) is substance specific.

• There are many methods employed, the two

most popular are:

– calibration curve (external standards),

– standard addition,

E t l t d d th d



•External standard method .Peak areas Ai are directly proportional to the amount of analyte injectedboth in standard (S) run:

and in sample (unknown, X) run:

Where:

F i is the proportionality constant (sensitivity of the detection-

integration system employed towards analyte i , tangent of a linearcalibration curve area vs. amount of analyte ) or the responsefactor,

V inj is the volume injected,

C i is the concentration of analyte in the original sample (our unknown).

F i can be determined from the first of the above equations and used in thesecond yielding:

and if volumes injected are equal (this is a condition difficult to achieve for

an inexperienced person):

A F V C iS i inj S iS ,

A F V C iX i inj X iX ,

C V C

A

A

V iX

inj S iS

iS

iX

inj X

,

,

C C A A

iX iS iX

iS

Standard addition method



Standard addition method .

Peak area A X1 in first injection (original sample) is:

and in second injection (after addition of m add the substance we want todetermine quantitatively):

F X and V inj assume the same values as in both equations, the latter if theanalytician is skilled, and C X2 is the concentration of unknown in the

sample after addition.Dividing both sides of the two equations yields:

We can also write:

The volume added in the denominator can be neglected, as it usuallyamounts to only a fraction of % of sample volume V S .

Substituting C X2 into the former equation and rearranging leads to final

expression:

A F V C X X in j X 1 1 1

A F V C X X in j X 2 2 2

A

A

C

C

X

X

X

X

1

2

1

2

C C V m

V V

C V m

V C

m

V X

X S add

S add

X S add

S

X add

S

21 1

1

C

A

A A

m

V X

X

X X

add

S 1

1

2 1



Standard addition is probably the best quantitative method. It allows forthe influence of the matrix on sensitivity (F X ), which can hardly vary in thetwo injections in the above procedure, but can differ significantly betweenthe samples and standard solutions used in the external calibration method. Also, it can easily compensate for irreproducibility of injection (a necessarycondition when using external standards). It makes the method particularlyattractive for inexperienced analyticians. To achieve the latter, the finalformula is :

Where CF is a correction factor:

where V inj2 and V inj1 are volumes injected before and after addition,respectively. We do not need to know them and they can be replaced byareas AR2 and AR1 or heights of a peak of selected reference substance,which is present in both injections at the same concentration. If theinjection volumes are identical, factor CF is equal to unity.

CF V

V

A

A

h

h

inj

inj

R

R

R

R

2

1

2

1

2

1

C A CF

A A CF

m

V X

X

X X

add

S 1

1

2 1



A relationship between the chromatographic parameters of a compound(t’ R,i) and its physico-chemical properties like a partition coefficients (K i).

where K A , K B are partition coefficients of analytes A and B, respectively,

k’ A , k’ B, their capacity factors , and a their ratios.

(The longer the component is retained in the column, the greater itscapacity factor.)

A B

k A

k B

K A

K B

,

'

'

k i

t R i

t M

t M

, ,



Mass spectrometry: “a technique for studying the masses of

atoms and molecules, or fragments of molecules. Gaseousspecies from condensed phase are ionized, the ions areaccelerated by an electric field and then separated accordingto their mass-to-charge ratio, m/z”.

lecture 7d- chromatography (1h) (2)

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